Method and kit for the prognostication of breast cancer

ABSTRACT

This invention relates to a method, kit and controls for detecting HER-2/neu gene amplification as a predictor of breast cancer reoccurrence and patient survival. The method is a fluorescent in-situ hybridization (FISH) assay using a labeled DNA probe. By determining the genetic nature of the cancer cells, appropriate treatment may be utilized. Control tumor cell lines with predefined amounts of HER-2/neu gene amplification are also disclosed.

[0001] This application is a continuation-in-part of U.S. provisionalpatent application Serial No. 60/072,574, filed Jan. 26, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to determining the prognosis of apatient with breast cancer by determining whether the HER-2/neu gene isamplified in tumor cells.

[0004] 2. Description of Related Art

[0005] Breast cancer remains a major cause of illness and death amongwomen in the United States, with over 180,000 new cases and 44,000deaths per year (American Cancer Society, 1997). Possibly the mostimportant predictor of clinical course in breast cancer is the presenceor absence of lymph node metastases. Many prognostic indicators aid inevaluation of invasive cancers in addition to the presence or absence oflymph node metastasis, including tumor size, histologic type, tumorgrade (differentiation reflected in extent of gland formation), nucleargrade (extent of nuclear alteration and frequency of mitosis), DNAcontent (ploidy), and hormone receptor status. A reasonable anddesirable approach would be the use of prognostic factors torisk-stratify invasive breast cancer patients into low-risk andhigh-risk groups in terms of the probability of recurrence (McGuire, etal., 1990).

[0006] The HER-2/neu (ERBB2) gene is an oncogene which sharessignificant homology to the epidermal growth factor receptor (EGFR) gene(Yamamoto, et al, 1986) and the retroviral gene v-erbB. It was firstdetected as a mutated transforming gene in chemically induced ratneuronal tumors. It has been isolated from diverse sources, including:rat neuroblastoma (Schechter et al, 1985); human tumor lines fromgastric cancer (Fukushige et al, 1986); salivary adenocarcinoma (Sembaet al, 1985); and a human breast cancer cell line where HER-2/neu wasidentified in an amplified form (King et al, 1985). The gene has beenlocalized to 17q11.2q12 (Human Gene Mapping 11, 1991), in a region whereseveral genes relevant to breast cancer are located, including BRCA1estradiol-17β dehydrogenase, NM23 and RARA.

[0007] Current evidence indicates that HER-2/neu protein over expressionand gene amplification are indicative of poor patient prognosis at allstages of breast tumor development. Amplification appears early in tumorprogression (Iglehart et al 1990 and Van de Vijver et al 1988), and whenpresent is homogeneously distributed throughout the tumor (Press et al,1994). Thus, it is a logical choice as a prognostic marker when used asan adjunct with other accepted prognostic indicators.

[0008] While such immunoassays for HER-2/neu protein have beencommercially available, interpreting results is somewhat difficult.Protein denaturation or degradation during handling, staining, embeddingin paraffin and sectioning gives variable results, including both falsenegatives and false positives. Additionally, slightly differentconditions during antibody-antigen binding results in false positivesand false negatives. Unacceptable results have been reported forimmunohistochemical detection of HER-2/neu amplification. See Thor et al1989; Richner et al, 1990; O'Reilly et al, 1991; and Lovekin et al,1991. By contrast, counting the number of copies of the HER-2/neu genein a cell represents a more objective determination and involves DNAmarkers which are less susceptible to degradation and provide lessvariable results.

[0009] HER-2/neu gene amplification status is useful as an adjunct inthe evaluation of the prognosis of node negative breast cancer patientsand is also an independent marker of high risk in node-negativepatients. Amplification of HER-2/neu is indicative of poor patientprognosis at all stages of breast cancer development and correlates withrelatively shorter disease-free and overall survival.

[0010] Studies have shown positive correlation between HER-2/neu geneamplification and other common indicators of poor prognosis in breastcancer (Tsuda, et al., 1989 and Seshadri, et al., 1993 and Slamon et al,U.S. Pat. No. 4,968,603). However, even strong breast cancer prognosticfactors, such as number of positive lymph nodes, tumor size andhistograde do not predict patient outcome unfalteringly (Wright, et al,1989 and Ro, et al., 1989). Current evidence indicates that HER-2/neuprotein over expression and gene amplification are indicative of poorpatient prognosis at all stages of breast cancer development (Seshadri,et al., 1993, Wright, et al., 1989 and Niehans et al., 1993). BecauseHER-2/neu amplification appears early in breast cancer progression(Iglehart, et al., 1990 and van de Vijver, et al, 1988) and, whenpresent is homogeneously distributed throughout the cancer (Inglehart,et al., 1990 and Press, et al., 1994), it can serve as a prognosticmarker for this disease (when used as an adjunct with other acceptedprognostic indicators).

[0011] The use of Fluorescent In-Situ Hybridization (FISH) targeted tothe HER-2/neu gene, has successfully demonstrated gene amplification inbreast cancer cell lines and primary tumors, and has shown that FISHresults are concordant with other measures of amplification(Kallioniemi, et al., 1992). [The gene has been localized to 17q11.2-q12(Human Gene Mapping 11, 1991), in a region where several genes relevantto breast cancer are located, including BRCA1, estradiol-17dehydrogenase, NM23, and RARA.] FISH technology combines the advantagesof direct gene amplification assessment with direct localization inmorphologically identified tumor cells. FISH is applicable to tumors ofall sizes because studies can be performed on sections from the originalspecimen blocks used for diagnosis. In many samples, direct comparisoncan be made with FISH assays on normal cells from the same preparation.Further, if amplification were localized rather than diffuselydistributed within a tumor, it would be detectable by FISH but could bediluted below detectable limits in extracted tumor DNA required forother procedures.

[0012] When performing an assay of such importance to the patient, it iscritical to have appropriate controls. Sections of previously testedtissue are somewhat undesirable as controls due to cell variability,unclear boarders, necrotic tissue in the center of the tumor, variableresponses to protease digestions and finite source material. Therefore,there is a need for quality control materials which can be run withevery test which lack the above mentioned problems.

[0013] There is also a need for a set of statistical benchmarks to allowthe medical practicioner to stratify the patient according to likelihoodof cancer recurrence. This will aid the practioner and patient indeciding whether agressive treatments (e.g. chemotherapy, radiation andanti-HER-2/neu therapy) should be empoloyed in lieu of a passive“watchful waiting” approach.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to methods and reagents whichdetermine the number of copies of the HER-2/neu gene in a breast cancerspecimen. This method uses a FISH assay for HER-2/neu in surgicallyremoved breast cancer tissue. Determination of an abnormally high copynumber of the gene correlates with poor prognosis and such patientsshould be treated aggressively.

[0015] The present invention is also directed to a set of controlslides, one of which has a normal copy number of the HER-2/neu gene, onehas a high copy number of the HER-2/neu gene and one has a slightlyelevated copy number of the HER-2/neu gene.

[0016] The present invention further includes the preparation of controlslides using cell lines instead of primary tumor tissue. The preferredcell lines used for controls are one with high amplification of theHER-2/neu gene, one with non-amplification and one with lowamplification.

[0017] The HER-2/neu gene detection system of the present invention is akit consisting of DNA probe and detection reagents that yields a greenfluorescent signal at the site of each HER-2/neu gene, on a bluefluorescent background of stained nuclear DNA. The kit is intended to beused with sections (4 μm) of formalin-fixed, paraffin-embedded humanbreast cancer tissue. The kit is untended to include or recommend theuse of another kit which includes the control lines.

[0018] The HER-2/neu gene detection system of the present invention ispreferably a fluorescence In situ hybridization (FISH) DNA probe assaythat determines the qualitative presence of HER-2/neu gene amplificationon formalin-fixed, paraffin-embedded human breast tissue as an aid tostratify breast cancer patients according to risk for recurrence ordisease-related death. It is indicated for use as an adjunct to existingclinical and pathologic information currently used as prognosticindicators in the risk stratification of breast cancer in patients whohave had a primary, invasive, localized breast carcinoma and who arelymph node-negative.

[0019] A recent review and comparison is Ross et al, The Oncologist 3:237-252 (1998).

BRIEF DESCRIPTION OF FIGURES

[0020]FIG. 1 is a survival curve of HER-2/neu Amplification Status withthe cumulative probability of early recurrence.

[0021]FIG. 2 is a survival curve of HER-2/neu Amplification Status withthe cumulative probability of disease-free survival.

[0022]FIG. 3 is a tumor size cumulative probability-1 cm overallsurvival

[0023]FIG. 4 is a an interaction, without error bars, of HER-2/neuamplification and tumor size cumulative probability of overall survivalHER-2/neu Amplification Status (amp+/amp−) and tumor size (>1 cm/<1 cm)

DETAILED DESCRIPTION OF THE INVENTION

[0024] The HER-2/neu gene amplification detection system according tothe present invention is a fluorescence in situ hybridization (FISH) DNAprobe assay intended for formalin fixed, paraffin-embedded human breasttissue as an aid in predicting risk of breast cancer recurrence so thatpatient management decisions can be improved. Post surgery lymph nodenegative patients with no amplification may receive little or no furthertreatment whereas patients with tumors having the HER-2/neu geneamplified may receive more aggressive monitoring, chemotherapy and/orradiation. Clearly, the appropriate use of the drugHERCEPTIN®(Genentech, South San Frisco, Calif.), humanized monoclonalantibody to HER-2/neu, is determinable by measuring HER-2/neu geneamplification. In the few months since release of the commercial assayit has become accepted standard practice to include the HER-2/neu geneamplification detection system routinely on breast cancer patients andparticularly before the particular therapy noted above.

[0025] The relationship between HER-2/neu gene amplification andprobability of remaining disease free and surviving is demonstrated inthe Figures. These clinical studies are based on breast cancer patientswho had excision of a primary, invasive, localized breast tumor, whowere node negative and who did not receive any adjuvant therapy exceptin cases of disease recurrence. While remaining disease-free is somewhatdifferent from survival, both measures are important even when the datadoes not exactly parallel.

[0026] While this specification is described with respect to breastcancer, one skilled in the art will readily appreciate the applicationof the techniques herein described of use with other cancers where theHER-2/neu gene is amplified, such as ovarian, prostate, endometrial andcertain colon cancers. In such situations, different control cell linesand different amplification cut off numbers may be required but the needfor appropriate quality controls remains.

[0027] Briefly, the methodology is as follows. Sections offormalin-fixed, paraffin-embedded breast cancer tissue mounted onmicroscope slides are pretreated chemically (Pretreatment Step,reduction of peptide disulfide bonds) and enzymatically (ProteinDigestion Step, digestion of proteins) to remove proteins that block DNAaccess. The DNA in the sections is converted from double- tosingle-strand by solution denaturation at 75° C. using a mixture of 20×SSC (saline sodium citrate) and formamide. A hybridization solution,containing labeled DNA probe which is complementary to the HER-2/neugene sequence, is applied to the tissue section, which is then incubatedunder conditions favorable for annealing of probe DNA and genomic DNAsequences. Unannealed probe is washed off using a mixture of 20× SSC andformamide. The hybridized probe is detected using a fluorescently-taggedligand (fluorescein-labeled avidin) which binds to the label on the DNAprobe, thereby immobilizing the fluorescein at the site of the HER-2/neugene. The remainder of the DNA is then stained with an intercalatingfluorescent counterstain (DAPI in Antifade). Excitement of fluoresceinand DAPI by light from a mercury arc lamp with appropriate filters in anepifluorescence microscope results in the emission of green and bluelight, respectively. The observer selects for these two colors by usinga microscope filter set designed for simultaneous viewing of DAPI andfluorescein, and scores nuclei in the tissue section for the number ofgreen signals on a blue background.

[0028] When performing such an assay of great importance to the patient,it is always necessary to use the best available controls. Tissuesections from an excised tumor are somewhat variable by being a mixedpopulation of cells, sometimes having unclear boarders. Tissue sectionscan only be as good at the tumor itself which may have a necroticcenter, have blood vessels through it, and contain a number ofinflammatory response cells. The density of cells in a primary tissuesection (tumor or normal) may be high with a 4 μm thick section havingonly a small part of a cell. In FISH assays, a protein digestion step isperformed. The digestion conditions differ between different tumors andnormal cell types. All of this requires skilled individuals to determinewhich cells are appropriate tumor cells to be considered on the slide.Additionally, tumor and normal sections represent a finite source ofcontrols as each new tissue block will require restandardization beforeit can become a control standard.

[0029] By comparison, cell lines have the advantage of uniformity incell type with no chance of misidentifying the cells. The cellconcentration is regulatable so that the control slide will have cellsevenly distributed and clearly separated. Since the cell line isuniform, the protein digestion conditions may be perfected, not merelyoptimized. Uniformity also reduces interpretation mistakes and permitsuse of less skilled, and less expensive, personnel. Because cell linesare used, the exact cell type may be used indefinitely as an permanentsource of control cells without further need to restandardize the cellline. Should doubt remain as to the advantages of standardized controlsover previously tested samples, several subclasses of U.S. patents aredevoted to analytical clinical controls, their preparation and use.

[0030] In the present invention, the control slides include a slide witha normal copy number of the HER-2/neu gene, a slide with a highlyamplified copy number of the HER-2/neu gene, and a slide with a lowlyamplified copy number of the HER-2/neu gene. Representative examplesare: Level 1 Control ATCC HTB 132 (MDA-MB-468) non-amplification, ≦3copies per cell Level 2 Control ATCC HTB 133 (T-47D) low amplification,3-10 copies per cell Level 3 Control ATCC HTB 30 (SK-BR-3) highamplification, ≧10 copies per cell

[0031] The use of a control with a low level of amplification ispreferred as clinical samples with low levels of gene amplification arethe mostly likely to be miss detected. Such primary cancers may bedifficult to find and standardize, thus the use of such a cell line hasconsiderable benefits.

[0032] It will be appreciated that numerous other cell lines may be usedas controls provided that the number of copies of HER-2/neu gene isadequately quantified and it is uniform in the cell line. Cell linecontrols should fall into one of the three level control ranges recitedabove. Once another cell line has been so standardized, it may be usedin lieu of the specific cell lines recited above. It is preferred to usetumor cell lines originating from the same tissue as being tested fromthe patient For example, the three cell lines above originated frombreast cancers.

[0033] Briefly, control slides are prepared by culturing the cell lines,suspending a predetermined concentration of cells in plasma, clottingthe plasma, formalin fixing, embedding in paraffin, sectioning andmounting on a slide. It should be noted that additional and alternativesteps of preparing the slide for FISH may also be performed with a goalof preparing the control cell line to resemble breast tumor tissue forcomparative parallel testing. Such sample preparation techniques aredescribed for example, in Diagnostic Molecular Pathology, Vol. 1, IRLPress, NY.

[0034] While these steps are individually well known in the art,numerous variations on the above procedure may be used. For example,other solidifying materials may be used in the place of plasma providedthat they do not alter the cellular DNA. Examples include agarose,gelatin, pectin, alginate, carrageenan, monomers, polymers etc. wherethe gel is formed by cooling, adding ions (calcium, potassium) adding apolymerizing or a cross linking agent, etc. Other fixatives are knownand may be used if any is desired at all. Paraffin embedding may bestandard but other similar materials may be used and may even beoptional. Likewise, the thickness of the section cut from a block isvariable and is optimized depending on the microscope and assayconditions.

[0035] The relative sensitivity and specificity of the HER-2/neu geneamplification detection system for measuring HER-2/neu gene copy numberswas accessed. Breast cancer specimens with a known HER-2/neu gene copyand expression levels were selected as archival tissue specimens.Amplification was previously determined by Southern Blot hybridizationor dot blot using extracted DNA. Expression had been determined byNorthern hybridization, Western immunoblotting and/orimmunohistochemistry using total RNA, total protein or histologicsections from tumor tissue. Slamon et al, 1989 and Press et al, 1993.Gene amplification levels correlated with gene expression levels inapproximately 90% of the breast cancers under research conditions. In aless standardized clinical setting, the divergence may be higher. Thecomparison with FISH was performed by the HER-2/neu gene amplificationgene detection system on 140 breast cancer specimens. Forty-nine wereconsidered true positive, 90 true negative, 0 false positive and 1 falsenegative. This is a the relative sensitivity value of 98% and therelatively specificity value of 100%.

[0036] The expected HER-2/neu gene detection system assay result innormal breast tissue (non-cancerous) was estimated in a population of 20breast tissue samples from reduction mammoplasties. The overall observedmean was 2.2 signals per nucleus with a range of 1.8-2.6 signals pernucleus. The target population for analysis using the HER-2/neu genedetection system was patients with primary node-negative, invasivebreast carcinoma. The expected prevalence of early recurrence within 2years is 4 to 6%. The expected prevalence of recurrence within 3 yearsis 2 to 10%. The expected prevalence of disease-related death (within 3years) is 10 to 15% (Clinical Oncology, 1993, page 207).

[0037] A clinical study evaluated HER-2/neu gene amplification status in220 women with node negative invasive breast cancer whose only course oftreatment was surgery, unless diagnosed with disease recurrence. Forthis study population HER-2/neu amplification was shown to havepredictive power independent of the other prognostic markers evaluated(patient age at diagnosis, tumor size, tumor grade, and estrogenreceptor). HER-2/neu was shown to be the strongest predictor for earlyrecurrence (within 24 months), recurrence and disease-related death.

[0038] The negative predictive value, probability of no disease beingpresent in women with HER-2/neu non amplified tumors, was found to behigh three years after diagnosis (93.3% based on a prevalence of 10.4%).The probability of being alive three years after diagnosis was 99.4%,based on a prevalence of 2.4%.

[0039] HER-2/neu was analyzed along with and controlling for the abovelisted prognostic factors. The combined effect (interaction) of tumorsize and HER-2/neu amplification status is presented in FIG. 4. Oneanalysis used tumor size at 1 cm (FIG. 3) and additional analysis lookedat tumor size at 2 cm. In both sets of analyses, tumor size is notsignificant (p>0.05) for predicting recurrence and disease-related deathwhen the tumor is HER-2/neu amplified.

[0040] When the tumor is not amplified for the HER-2/neu gene, tumorsize is also an insignificant predictor of recurrence anddisease-related death within 3 years. With longer follow-up,disease-related death was significantly predicted in a comparison oftumors>1 cm. In this particular data set, there were no disease-relateddeaths for HER-2/neu non-amplified tumors.

[0041] These data show that for this study tumor size failed to be agood predictor of recurrence and disease-related death within 3 years.Tumor size is of little consequence in HER-2/neu positive tumors andonly becomes of value when evaluating disease-related death, inHER-2/neu negative tumors . HER-2/neu amplification was shown to havepredictive power independent of all other prognostic markers evaluatedand to be the strongest predictor for recurrence and disease-relateddeath.

[0042] The following Examples utilized the commercially available OncorINFORM HER-2/neu Gene Detection System (Ventana Medical Systems,Gaithersburg, Md., USA), Cat. No: S8000-KIT. The Procedure andInterpretation Guide enclosed with the kit is expressly incorporated byreference. This kit was the subject of a FDA PMA No. P9400004, thepublic contents of which are expressly incorporated by reference.

EXAMPLE I Preparation of HER-2/neu Probes

[0043] Partial restriction enzyme digests of human Chromosome 17 DNAwere prepared to create a library. Fragments were cloned into BAM H1restriction sites on a cosmid vector and grown in E. Coli HB 101.Positive clones were selected with Kanamycin containing medium. Thecosmid probe set represent overlapping segments with a four membercontig. A probe used to detect the cDNA is preparable using primers5′-CGGCCAAGATCCGGGAGTTGGT-3′ and 5′-TCTTGATGCCAGCAGAAGTCAGGC-3′.Numerous publications exist regarding the HER-2/neu gene and otherprobes may be prepared and used.

[0044] Biotintylated HER-2/neu DNA probe was prepared containing abiotin-labeled single-stranded DNA fragment derived from human genomicDNA sequences, suspended in a solution of formamide, SSC and blockingDNA. The probe DNA sequences are complementary to the sample HER-2/neu(erb-b2) gene sequence and specifically bind to them upon hybridization.The probe used below is the commercially available HER-2/neu probe(Ventana Medical Systems, Gaithersburg, Md.)

EXAMPLE II Sample Preparation for Fish Assay for HER-2/neu

[0045] Slides were prepared by cutting paraffin embedded tissue samplesinto 4 μm thin sections and applying them to silanized or positivelycharged slides. The slides were air dried and baked at 65° C.±5° C.overnight. The slides were deparaffinized in fresh xylene that has notbeen used for more than one week and repeated through three changes ofxylene for five minutes each. The slides were then washed in fresh 100%ethanol that has not been used for more than one week for two minutes.The ethanol washing was repeated and the slides allowed to air dry.

[0046] The slides were pretreated by immersing slides in a coplin jarcontaining 40 ml of pre-warmed 30% w/v sodium bisulfite PretreatmentSolution in a 43° C.±2° C. water bath for 15 minutes. This solution isdesigned to reduce disulfide bonds, aid in protein digestion and improveprobe penetration to target DNA sequences. The slides were then washedin 40 ml of 2× SSC at room temperature for 1 minute and then washedtwice using fresh 2× SSC. The slides were then dehydrated through aroom-temperature graded series of ethanol solutions for 2 minutes ineach of 70%, 80%, 90% and 100% ethanol and allowed to air dry inclinedwith label end down.

[0047] 40 ml of Protein Digesting Enzyme Working Solution was freshlyprepared by mixing 25 mg of proteinase K in 37° C.±2° C. of prewarmed 2×SSC. Slides were immersed in a coplin jar of prewarmed Protein DigestingEnzyme Working Solution and incubated at 37° C.±2° C. for 40 minutes.This solution is needed to digest protein and improve probe penetration.The slides were washed three times in 40 ml of fresh 2× SSC at roomtemperature for 1 minute. The slides were dehydrated through theroom-temperature graded series of ethanol solutions for 2 minutes ineach grade of ethanol: 70%, 80%, 90% and 100% and allowed to air dryinclined with label end down.

[0048] The slides were denatured by immersing them in a coplin jarcontaining 40 ml of prewarmed Denaturation Solution (70% formamide/2×SSC, pH 7.0) in a 75° C. water bath for 8 minutes. The slides wereimmediately transferred to the pre-chilled (−20° C.±5° C.) 70% ethanoland rinsed for 2 minutes. The rinse was repeated in pre-chilled (−20°C.±5° C.) 80%, 90%, and 100% ethanol solutions, successively and allowedto air dry inclined with label end down.

[0049] The HER-2/neu DNA probe was prewarmed at 37° C.±2° C. for 5minutes, vortexed for 1 minute and centrifuged for 2 to 3 seconds tocollect contents in the bottom of the tube. 10 μl of probe solution waspipetted onto the denatured tissue section and covered gently with a 25mm×25 mm glass coverslip. Larger tissue sections may require up to 20 μlof probe and larger glass coverslips. The slides were incubated at 37°C.±2° C. for 12 to 16 hours in a humidified chamber.

[0050] The coverslips were then removed by sliding it to the side andlifting the overhanging edge with forceps. The slides were washed in acoplin jar containing 40 ml of pre-warmed Post-Hybridization WashSolution (50% Formamide/2× SSC, pH 7.0) in the 43° C.±°C. water bath for15 minutes. The slides were then rinsed in a coplin jar containing 40 mlof pre-warmed 2× SSC in the 37° C. water bath with frequent agitationfor 10 minutes and repeated with fresh 2× SSC and placed in a coplin jarcontaining 40 ml of 1× PBD (phosphate buffered detergent) at 18° C. to25° C.

[0051] 60 μl of Blocking Reagent One (0.05 g nonfat dry milk, NonidetP40, phosphate buffer and sodium azide) was added to each slide, aplastic coverslip placed over the solution and incubated 5 minutes in ahumidified chamber at room temperature. The plastic coverslip was pealedoff and blotted dry for re-use. The slide was tilted to allow fluid todrain briefly. This reagent contains salts, detergent, proteins andsodium azide (preservative) which aid in reducing non-specific bindingof fluorescein-labeled avidin to the hybridized and washed tissuesection.

[0052] 60 μl of Detection Reagent (fluorescein labeled avidin in sodiumazide preservative) was added to each slide and the plastic coverslipreplaced over the solution. The slide was incubated 20 minutes in ahumidified chamber at room temperature. After 10 minutes of the 20minute incubation, one lifts and replaces the plastic coverslip toensure even fluid distribution. This reagent detects hybridized probeDNA by binding to the biotin conjugated to the probe.

[0053] The plastic coverslip was then pealed off and discarded. Theslides were washed in a coplin jar containing 40 ml of 1× PBD at roomtemperature for 2 minutes and the wash repeated 2 times using fresh 1×PBD.

[0054] The slides were removed from 1× PBD, tilted to allow fluid todrain briefly, then excess fluid was briefly blotted from the edge.

[0055] 60 μl of Blocking Reagent Two (0.05 ml goat serum, Nonidet P40,phosphate buffer and sodium azide) was added to each slide and a freshplastic coverslip placed over the solution. The slides were incubated 5minutes in a humidified chamber at room temperature, the plasticcoverslip pealed off and blotted dry for reuse and the slide tilted toallow fluid to drain briefly. This reagent is a mixture of salts,detergent and proteins in a sodium azide preservative which reducenon-specific binding of the Anti-Avidin Antibody to the hybridized answashed tissue section during the signal amplification phase ofdetection.

[0056] 60 μl of Biotin-labeled Anti-Avidin Antibody was added to eachslide and the plastic coverslip replaced over the solution. Incubationwas for 20 minutes in a humidified chamber at room temperature. At 10minutes of incubation, the plastic coverslip was lifted and replaced toensure even fluid distribution. This reagent binds tofluorescein-labeled avidin what has previously bound to the hybridizedprobe and allows amplification of the fluorescent signal by providingmultiple additional biotin moieties for binding by fluorescein-labeledavidin for each one originally bound with probe. The reagent containssodium azide as a preservative.

[0057] The plastic coverslip was removed and discarded. Slides werewashed in a coplin jar containing 40 μl of 1× PBD at room temperaturefor 2 minutes. This wash was repeated 2 times using fresh 1× PBD.

[0058] 60 μl of Blocking Reagent One was applied to each slide and afresh plastic coverslip placed over the solution. Incubation was for 5minutes in a humidified chamber at room temperature. After that theplastic coverslip was pealed and blotted dry for reuse while the slidewas tilted to allow fluid to drain briefly.

[0059] 60 μl of Detection Reagent was applied to each slide and theplastic coverslip replaced over the solution. Incubation was for 20minutes in a humidified chamber at room temperature. After 10 minutes ofincubation, the plastic coverslip was lifted and replaced to ensure evenfluid distribution.

[0060] The plastic coverslip was pealed and discarded and the slideswashed in a coplin jar containing 40 ml of 1× PBD at room temperaturefor 2 minutes. The wash was repeated 2 times using fresh 1× PBD.

[0061] The cell nuclei were counterstained by adding 20 μl ofDAPI/Antifade (DAPI, glycerol, P-phenylene diamine dihydrochloride,sodium bicarbonate, sodium hydroxide in phosphate buffered saline) toeach slide and covered with a 24×50 mm glass coverslip. Stained slidesmay be stored in the dark at −15° C. to −25° C. for up to five daysbefore analysis. This reagent is a mixture of a blue-fluorescingDNA-intercalating dye and a chemical which reduces photo bleaching. Thisis used to counterstain nuclear DNA blue to prolong probe signalfluorescence.

[0062] If the tissue section was insufficiently digested under thedesignated digestion conditions and is determined to interfere withinterpretation of assay results, an extended protein digestion may beused as follows. The coverslip was removed by gently wiping off theimmersion oil with tissue paper and soaking the slide in 40 ml 2× SSC,pH 7.0 in a coplin jar at room temperature until the coverslip fallsoff. The slide was placed in a coplin jar containing fresh 2× SSC, pH7.0 for several minutes to clean off any residual DAPI/Antifade.

[0063] The slides were placed in prewarmed Protein Digestion EnzymeWorking Solution at 37° C.±2° C. The effect of this protein digestionand the initial digestion is cumulative. Twenty (20) additional minutesof digestion might be an appropriate starting time for tissue that seemsvery undigested after the initial 40 minute digestion.

[0064] The slides were washed in 40 ml 2× SSC, pH 7.0 room temperaturewith agitation for 10 seconds, then dehydrated in 70%, 80%, 90%, and100% ethanol at room temperature for 1 minute each and allowed to airdry. The process above is then repeated.

Example III Preparation of Control Slides

[0065] The control slides are prepared using the following cell lines:

[0066] Level 1 Control ATCC HTB 132 (MDA-MB-468) cell line

[0067] Level 2 Control ATCC HTB 133 (T-47D) cell line

[0068] Level 3 Control ATCC HTB 30 (SK-BR-3) cell line

[0069] Each cell line is available from the American Type CultureCollection, Manassas, Va., USA and grown using standard media andtechniques to produce approximately 1.5×10⁸ cells. The cell growth isdivided into approximately 30 5×10⁶ cells. Pellets of the cultured cellswere suspended in a plasma/thrombin matrix and clotted. Fibrin clottedblocks were then formalin fixed. Each block is paraflin embedded, cutinto 4 μm sections and mounted on a silanized glass slide. The HER-2/neuControl Slides were stored at 18° C. to 25° C. prior to processing andat −15° C. to −25° C. after processing

EXAMPLE IV Scoring of Control Slides

[0070] A non-amplified sample has a mean HER-2/neu signal per nucleusless than or equal to (≦) 4. Specimens with a mean signal per nucleusgreater than (>) 4 are amplified for the HER-2/neu gene. Control Slidesare divided into three (3) categories. These are listed below and shouldnot be confused with the amplification cut-off value of 4.

[0071] Level 1 (0 to 3 Signals/Nucleus) Control Slides

[0072] A Level 1 control has a mean signal per nucleus value of lessthan or equal to 3. This range of assay scores (0 to 3) is defined asnon-amplified for the HER-2/neu gene. In most patient specimens, aninternal non-amplified control is present in the form of cells that areidentifiably non-cancerous by the criteria of histopathologicmorphology.

[0073] Level 1 control slides are 4μm sections of a formalin-fixed,paraffin-embedded human breast cancer tissue culture cell line onsilanized slides. The preferred cell line is MDA-MB-468 (ATCC # HTB132).

[0074] Level 2 (>3 to <10 Signals/Nucleus) Control Slides

[0075] A Level 2 control has a mean signal per nucleus value of greaterthan 3 to less than 10. This range of assay scores (>3 to <10) isdefined as low amplified HER-2/neu gene amplification. Level 2 controlslides are 4μm sections of a formalin-fixed, paraffin-embedded humanbreast cancer tissue culture cell line on silanized slides. Thepreferred cell line is T-47D (ATCC # HTB 133).

[0076] Level 3 (Symbol≧10 Signals/Nucleus) Control Slides

[0077] Level 3 control specimens represent a highly amplified specimen.A Level 3 control has a mean signal per nucleus value equal to orgreater than 10. This range of assay scores (≧10) is well above thecutoff of >4 signals per nucleus. Level 3 control slides are 4 μmsections of a formalin-fixed, paraffin-embedded human breast cancertissue culture cell line on silanized slides. The preferred cell line isSK-BR-3 (ATCC # HTB 30).

[0078] Level 1, Level 2 and Level 3 controls should be run and evaluatedwith each run of the HBER-2/neu gene detection system assay.Paraffin-embedded human breast cancer cell lines are run simultaneouslywith each run of samples. The control slides are read by scoring 20cells from each of two (2) randomly selected areas of the slide (totalof 40 nuclei) and the results interpreted as described below. Scoringcriteria for invasive cancer do not apply to the paraffin-embedded cellline controls.

[0079] Because breast cancer cell nuclei are often considerably thickerthan the 4 μm sections of tissue required to perform the assay, thecontrol tissue nuclei are frequently not intact. This effect ofsectioning will result in the observation of fewer HER-2/neu signalsthan are actually contained in an intact nucleus.

[0080] The mean signal per nucleus of a Level 1 control must be lessthan the mean signal per nucleus of a Level 2 control for a processingrun to be considered valid. For cell line controls acceptance ranges seethe list below:

[0081] Based on 393 observations (40 nuclei scored per observation) of 4μm sections of the Level I control cell line a mean of 2.4 (standarddeviation=0.25) HER-2/neu signals per nucleus was determined.

[0082] Based on 102 observations (40 nuclei scored per observation) of 4μm sections of the Level 2 control cell line, a mean of 3.5 (standarddeviation=0.71) HER-2/neu signals per nucleus was determined.

[0083] Based on 338 observations (40 nuclei scored per observation) of 4μm sections of the recommended Level 3 control cell line, an acceptancerange of 15.8 to 20.0 HER-2/neu signals per nucleus was determined(determined by non-parametric analysis).

[0084] In addition to these HER-2/neu Control Slides, controls may alsotake the form of 4 μm tissue sections from invasive breast cancers thathave been previously identified to have specific levels of HER-2/neugene amplification by fluorescence in situ hybridization (FISH). Use ofbreast cancer tissue as control material requires qualification andvalidation by the user laboratory according to the laboratory'sestablished procedures. While these controls may be useful to classifyor group tissues according to HER-2/neu amplification status suchcontrols are time consuming to prepare and generally considered inferiorto standardized controls. Additionally, controls from cell lines arehomogeneous and reproducible, neither quality can be attributed tosurgically removed tumors and their sections

[0085] For determination of HER-2/neu gene amplification level in tissuespecimens, 40 nuclei were scored from specimens processed with two (2)lots of the control cell lines. Multiple observers were used (3 or 4) toachieve accurate estimates of the mean and standard deviation (SD).Acceptance ranges were calculated from the mean plus and minus three (3)standard deviations. The results of six (6) Level 1 tissue specimens aresummarized below. TABLE 1 Examples of Level 1 Tissue Specimen Means andAcceptance Ranges Specimen Mean ± SD, ( no. of observations) AcceptanceRange  1. 1.96 ± 0.37, (N = 42) 0.85-3.07 2 2.14 ± 0.79, (N = 42)  0-4.51 3 2.01 ± 0.41, (N = 42) 0.78-3.24 4 2.08 ± 0.64, (N = 42)0.16-4.00 5 1.70 ± 0.31, (N = 36) 0.77-2.63 6 2.01 ± 0.29, (N = 36)1.14-2.88

[0086] In general, a 4 μm section of a Level 2 control tissue willexhibit a mean of greater than 3 to less than 10 (>3 to <10) HER-2/neusignals per nucleus (40 nuclei scored) when assayed with the HER-2/neugene detection system of the present invention.

[0087] For determination of HER-2/neu gene amplification level in tissuespecimens, 40 nuclei were scored from specimens processed with two (2)lots of the control slides. Multiple observers were used (3 or 4) toachieve accurate estimates of the mean and standard deviation (SD).Acceptance ranges were calculated from the mean plus and minus two (2)standard deviations. The results of ten (10) Level 2 tissue specimensare summarized below. TABLE 2 Examples of Level 2 Tissue Specimen Meansand Acceptance Ranges Specimen Mean ± SD, ( no. of observations)Acceptance Range 1 3.93 ± 0.74, (N = 36) 2.46-5.41 2 5.69 ± 1.31, (N =36) 3.07-8.31 3 3.97 ± 1.65, (N = 36) 0.67-7.27 4 3.56 ± 0.47, (N = 36)2.62-4.50 5 3.05 ± 0.41, (N = 36) 2.23-3.87 6 6.35 ± 1.06, (N = 51)4.23-8.47 7 8.07 ± 2.08, (N = 53)  3.91-12.23 8 6.17 ± 2.19, (N = 36) 1.79-10.55 9 5.52 ± 1.6, (N = 36)  2.32-5.52 10 8.21 ± 2.47, (N = 30) 3.27-13.51

[0088] In general, a 4 μm section of a Level 3 control tissue willexhibit a mean of greater than or equal to 10 HER-2/neu signals pernucleus (40 nuclei scored) when assayed with the HER-2/neu genedetection system.

[0089] In a study for determination of HER-2/neu gene amplificationlevel in tissue specimens, 40 nuclei were scored from specimensprocessed with two (2) lots of the control slides. Multiple observerswere used (3 or 4) to achieve accurate estimates of the mean. Acceptanceranges were calculated from the mean plus and minus two (2) standarddeviations with the upper limit truncated at 20. The results of eight(8) Level 3 tissue specimens are summarized below. TABLE 3 Examples ofLevel 3 Tissue Specimen Means and Acceptance Ranges Specimen Mean ( no.of observations) Acceptance Range 1 17.72 (N = 36) 12.34-17.72  2 17.46(N = 36) 11.66-20.00* 3 15.95 (N = 36) 11.15-20.00  4 10.85 (N = 44)6.25-15.45 5 14.54 (N = 42) 8.84-20.00 6 10.73 (N = 42) 6.16-15.31 715.09 (N = 36) 6.19-20.00 8 12.20 (N = 36) 4.46-19.94

EXAMPLE V Fish Assay for HER-2/neu Amplification on Clinical Samples

[0090] Slides were viewed with an epifluorescence microscope equippedwith a DAPI filter set and a DAPI/FITC/Texas Red triple band pass filterset (a filter set capable of simultaneously passing FITC and DAPIfluorescence). A FITC/Texas Red dual band pass filter set (a filter setthat allows visualization of the FITC signal but not the DAPIcounterstain) is helpful in resolving background from true signal. Themicroscope may be equipped with 10×, 40× (optional for viewinghematoxylin and eosin stained sections) and 100× objectives and a 100watt mercury arc light source. Scoring should be performed in a darkenedroom with excessive light leaking from microscopes minimized.

[0091] Using the DAPI filter set and the same low power objective usedto view hematoxylin and eosin stained sections, it was confirmed thatthe tissue section contains areas of invasion as previously identifiedin hematoxylin and eosin stained sections. Areas of invasion are scored;carcinoma in situ should not be scored.

[0092] Using the DAPI/FITC/Texas Red triple band pass filter set and a100× oil objective, the FITC signal was present in approximately ¾ ormore of the cancer cell nuclei in the area to be scored.

[0093] It should be noted that non-cancerous cell nuclei (e.g. fromnormal epithelium) may be more resistant to protein digestion and mayshow lower levels of hybridization than tumor cell nuclei; therefore,these non-cancerous cell nuclei are not a reliable gauge ofhybridization efficiency for the cancerous cell nuclei. Thehybridization signals to be scored within a cancer cell nucleus will beof similar size and intensity, whether separated or clustered.

[0094] With the DAPI filter set and a 100× oil objective, individualcancer nuclei were selected for scoring. Only cancer nuclei that arenon-overlapping are selected. Severely truncated cancer nuclei wereexcluded. Cancer nuclei that are less than ⅓ the diameter of the averagecancer cell nucleus are not selected. Overdigested and mechanicallydamaged cancer cell nuclei are not selected. Only cancer cell nucleithat have relatively well-defined borders are selected to be scored.

[0095] Using the DAPI/FITC/Texas Red triple band pass filter set and a100× oil objective, probe signals were differentiated from background ifpresent. FITC stain appearing over cytoplasm or in the extra-cellularmatrix is considered background. Background confined to the cancernucleus is more difficult to interpret and could interfere withcounting, but the background is generally much smaller and more diffusethan true probe signal.

[0096] Using the DAPI/FITC/Texas Red triple band pass filter set and a100× oil objective, the number of FITC signals present in each of 20randomly-selected cancer nuclei that meet all the above mentionedcriteria were counted. In FISH analysis, signals are often in differentplanes of focus within the tumor cell nucleus. Focusing up and downthrough the section to find all of the signals present in the cancercell nucleus was used. If the signal count is greater than 20 per cancercell nucleus, it was recorded as 20+ and not grouped with any othercounts.

[0097] Scoring in a second area of invasive breast cancer was repeatedfollowing all steps above. The two areas examined were separate,distinct microscopic areas within a single section. The total number ofcancer nuclei scored was 40 from 2 distinct areas of the same lesion inone section the mean number of HER-2/neu signals per nucleus wasdetermined.

[0098] If more than 5% of the fluorescein signals (those of similar sizeand intensity to true signal within invasive tumor nuclei) are locatedover the cytoplasmic compartment or extra cellular matrix and alltroubleshooting methods have been exhausted, the background is excessiveand the assay repeated.

[0099] When the positive or negative control results fall outside theexpected values, then the specimen results are unreliable and the assayrepeated.

[0100] When 40 non-overlapping nuclei cannot be identified, then thesample is inadequate and the assay repeated on a new slide.

[0101] When signal intensity varies widely after all troubleshootingmethods have been exhausted, then the specimen results is unreliable andthe assay repeated.

[0102] Values at or near the cut-off (3.5 to 4.5 mean signals/nucleus)are expected to occur in approximately 3.6% of the patient population.Scoring of borderline specimens should be repeated by another qualifieduser or the test should be repeated using a new tissue section. If thevalue of 3.5 to 4.5 persists, then the borderline results should beinterpreted with caution and increased emphasis should be given to theother clinical and prognostic information available to the practitioner.

[0103] A retrospective study of 220 node-negative breast cancer patientspecimens were collected from multiple sources and analyzed at twoclinical sites in the United States. This combined data set was used todetermine the association of HER-2/neu gene amplification, using theHER-2/neu gene detection system of the present invention, to theclinical outcomes; early recurrence (within 24 month of diagnosis),recurrences, and death, due to breast cancer.

[0104] The clinical performance characteristics of the HER-2/neu genedetection system are described with amplification defined as >4 signalsper nucleus and non-amplification defined as <4 signals per nucleus.

[0105] The HER-2/neu gene detection system was used to retrospectivelyidentify the risk of recurrence and death for node-negative breastcancer patients meeting the following criteria:

[0106] 1) Diagnosis of invasive breast cancer;

[0107] 2) Available formalin-fixed, paraffin-embedded tissue forHER-2/neu analysis;

[0108] 3) Primary treatment surgery only;

[0109] 4) Clinical follow up for at least 2 years for early recurrence,3 years for recurrence and death.

[0110] The safety and effectiveness of the HER-2/neu Gene DetectionSystem was evaluated in a population of 220 node-negative, invasivebreast cancer patients for early recurrence within two years. Twohundred twelve (212) of the 220 specimens were eligible for evaluationof recurrence at anytime and 210 of the 220 specimens were eligible forevaluation of disease-related death. Eight (8) subjects did not recurand were lost to follow-up before 36 months; ten (10) subjects did notdie of their disease and were lost to follow-up before 36 months.) Therelationship of the HER-2/neu gene detection system assay result to theprobability of remaining recurrence-free (disease-free survival) inlymph node negative breast cancer is presented in Table 4. Therelationship of the assay results to the probability of surviving(overall survival) is shown in Table 5.

[0111] The survival curves presented in the figures are graphicalrepresentations of the probability of early recurrence-free survival (norecurrence within 24 months) (FIG. 1), recurrence-free survival (norecurrence at anytime) (FIG. 2), and overall survival for subjects withand without HER-2/neu amplification. Error bars show the standard erroraround the values. TABLE 4 Probability of disease-free survival ofbreast cancer patients with non-amplified and amplified lesions. Timefrom Probability of Remaining Disease-Free* Surgery Non-AmplifiedAmplified (in Years) (95% CI)† N** (95% CI)† N** 0.5  100% (100.0% to100.0%) 179 93.8% (85.7% to 100.0%) 31 1.0 98.3% (96.4% to 100.0%) 17681.8% (68.7% to 95.0%) 27 1.5 96.7% (94.1% to 99.2%) 173 75.8% (61.1% to90.5%) 25 2.0 94.4% (91.1% to 97.7%) 169 75.8% (61.1% to 90.5%) 25 2.593.9% (90.3% to 97.4%) 168 72.7% (57.4% to 88.0%) 24 3.0 93.3% (89.6% to97.0%) 167 69.7% (54.0% to 85.4%) 23 5.0 85.9% (80.6% to 91.2%) 12166.7% (50.6% to 82.7%) 19 10.0 70.5% (60.3% to 80.7%) 23 61.9% (44.5% to79.3%) 4 Non-Amplified Amplified Time from Cumulative Probability ofCumulative Probability of Surgery Cumulative No. Cases RemainingCumulative No. Cases Remaining (in years) N** No. Events CensoredDisease Free N** No. Events Censored Disease Free 0.5 179 0 0 100.0%  312 0 93.9% 1.0 176 3 0 98.3% 27 6 0 81.8% 1.5 173 6 0 96.7% 25 8 0 75.8%2.0 169 10 0 94.4% 25 8 0 75.8% 2.5 168 11 0 93.9% 24 9 0 72.7% 3.0 16712 0 93.3% 23 10 0 69.7% 5.0 121 24 34 85.9% 19 11 3 66.7% 10.0 23 35121 70.5% 4 12 17 61.9%

[0112] TABLE 5 Probability of Overall Survival Tumor Size large (>1 cm)/ small (<1 cm) and HER-2/neu Amplification Status Probability ofoverall survival of breast cancer patients with large/small and non-amplified/amplified tumors. Probability of Survival* Small (≦1 cm);Small (≦1 cm); Time from Surgery Non-Amplified (≦4) Amplified (>4) (inYears) (95% CI)† N** (95% CI)† N** 0.5  100% (100.0% to 100.0%) 39  100%(100.0% to 100.0%) 5 1.0  100% (100.0% to 100.0%) 39  100% (100.0% to100.0%) 5 1.5  100% (100.0% to 100.0%) 39  100% (100.0% to 100.0%) 5 2.0 100% (100.0% to 100.0%) 39 80.0% (44.9% to 100.0%) 4 2.5  100% (100.0%to 100.0%) 39 80.0% (44.9% to 100.0%) 4 3.0  100% (100.0% to 100.0%) 3980.0% (44.9% to 100.0%) 4 5.0  100% (100.0% to 100.0%) 29 60.0% (17.1%to 100.0%) 2 10.0  100% (100.0% to 100.0%) 6 60.0% (17.1% to 100.0%) 1Probability of Survival* Large (>1 cm;) Large (>1 cm); Time from SurgeryNon-Amplified (≦4) Amplified (>4) (in Years) (95% CI)† N** (95% CI)† N**0.5  100% (100.0% to 100.0%) 117  100% (100.0% to 100.0%) 25 1.0  100%(100.0% to 100.0%) 117  100% (100.0% to 100.0%) 25 1.5  100% (100.0% to100.0%) 117 96.0% (88.4% to 100.0%) 24 2.0  100% (100.0% to 100.0%) 11796.0% (88.4% to 100.0%) 24 2.5 99.2% (97.4% to 100.0%) 116 96.0% (88.4%to 100.0%) 24 3.0 99.2% (97.4% to 100.0%) 116 88.0% (75.3% to 100.0%) 225.0 96.4% (92.9% to 99.9%) 89 68.0% (49.8% to 86.2%) 15 10.0 83.4%(74.2% to 92.6%) 21 51.0% (19.1% to 82.9%) 3 # (death or recurrence) orbeing lost to follow-up.

EXAMPLE VI Multi-Tiered Cutoffs for HER-2/Neu Gene Copy

[0113] The data from the above testing was analyzed to determine theeffect for using a ≦3 cutoff and a ≦10 cutoff on early recurrence(within 24 months), recurrence anytime and disease related death at anytime. The relative hazard for each was calculated buth unadjusted andadjusted for estrogen receptor, tumor size, patient age, study site andtumor grade. The results are in Tables 6. TABLE 6 Relative RiskUnadjusted Adjusted ≦3 cutoff ≦10 cutoff ≧10 ≦3 cutoff ≦10 cutoff ≧10Early Recurrence 4.8 6.6 7.8 4.3 5.5 8.3 Recurrence 2.0 3.4 3.4 2.0 3.84.3 Death 4.7 5.8 6.9 4.5 7.3 11.0

[0114] As can be seen from this data, the higher the average HER-2/neugene copy number per cell, the greater the risk to the patient.

[0115] Although preferred embodiments are specifically described herein,it will be appreciated that many modifications and variations of thepresent invention are possible in light of the above teachings andwithin the purview of the appended claims without departing from thespirit and intended scope of the invention. Other and furtherembodiments will be apparent to those in the art from the precedingdescription and examples. No unreasonable limitations or the like are tobe drawn therefrom in interpreting the following claims.

[0116] References cited in text and related to the invention.

[0117] American Cancer Society (ACS), 1997. Cancer facts and figures;Atlanta Ga.: The American Cancer Society.

[0118] Clinical Oncology: A Multidisiplinary Approach for Physicians andStudents , 7th Edition. (Editor P. Rubin, M.D.), W.B. Saunders Company,Philadelphia, Pa., 1993, pg. 207.

[0119] Cox, D. R., 1972. Regression models and life-tables (withdiscussion); Journal of the Royal Statistical Society, B, 34: 187-220.

[0120] Glick, J., 1988. Meeting highlights: Adjuvant therapy for breastcancer; Journal of the National Cancer Institute, 80:471-475.

[0121] Greenwood, M., 1926. The natural duration of cancer. Reports onPublic Health and Medical Subjects, Vol. 3, Her Majesty's StationaryOffice, London: 1-26.

[0122] Human Gene Mapping 11, 1991. London Conference (1991) EleventhInternational Workshop on Human Gene Mapping; Cytogenetics and CellGenetics, Solomon, E., Rawlings, C., eds., Karger, 58:1-4:702.

[0123] Iglehart, J. D., Kraus, M. H., Langton, B. C., Huper, G., Kems,B. J., Marks, J. R., 1990. Increased erbB-2 gene copies and expressionin multiple stages of breast cancer; Cancer Res., 50:6701-6707.

[0124] Kaplan, E. L., & Meier, P. L., 1958: Non-parametric Estimationfrom Incomplete Observations; Journal of the American StatisticalAssociation, 53:447-491.

[0125] Kallioniemi, O. P., Kallioniemi, A., Kurisu, W., Thor, A., Chen,L.C., Smith, H. S., Waldman, F. M., Pinkel, D., Gray, J. W., 1992. ERBB2amplification in breast cancer analyzed by fluorescence in situhybridization; Proc. Natl. Acad. Sci. USA, 89:12:5321-5325.

[0126] McGuire, W., Tandon, A., Allred, C., Chamnes, G., Clark, G.,1990. How to use prognostic factors in axillary node-negative breastcancer patients; J. Natl. Cancer Inst., 82:12:1006-1015.

[0127] National Cancer Institute (NCI), Clinical Alert from the NationalCancer Institute, Department of Health and Human Services, Bethesda,Md., May 16, 1988.

[0128] Niehans, G. A., Singleton, T. P., Dykoski, D., Kiang, D. T.,1993. Stability of HER-2/neu expression over time and at multiplemetastatic sites; J. Natl Cancer Inst, 85:15:1230-1235.

[0129] Press, M. F., Hung, G., Godolphin W., Slamon, D. J., 1994.Sensitivity of HER-2/neu antibodies in archival tissue samples:Evaluation of immunostaining in multi-tumor tissue blocks of breastcancers with molecularly characterized application and expressionlevels; Cancer Res., 54:2771-2777.

[0130] Ro, J., El-Naggar, A., Ro, J., et al., 1989. c-erbB-2amplification in node negative human breast cancer; Cancer Research,49:6941.

[0131] Seshadri, R, Firgaria, F. A., Horsfall, D. J., McCaul, K.,Setlur, V., Kitchen, P., 1993. Clinical significance of HER-2/neuoncogene amplification in primary breast cancer; Journal of Clin.Oncology, 11:10:1936-1942.

[0132] Tsuda, H., Hirohashi, S., Shimosato, Y., Hirota, T., Tsugane, S.,Yamamoto, H., Miyajima, N., Toyoshima, K., Yamamoto, T., Yokota, J.,Yoshida, T., Sakamoto, H., Terada, M., Sugimura, T., 1989. Correlationbetween long-term survival in breast cancer patients and amplificationof two putative oncogene coamplification units; hst-1/int-2 andc-erbB-2/ear-1; Cancer Res., 49:3104-3108.

[0133] Van de Vijver, M. J., Peterse, J. L., Mooi, W. J., et al., 1988.Neu-Protein overexpression in breast cancer. Association withcomedo-type ductal carcinoma in situ and limited prognostic value instage II breast cancer, N. Engl. J. Med., 319:1239-1245.

[0134] Wright, C., Nicholson, S., et al., 1989. Expression of c-erbB-2oncoprotein: a prognostic indicator in human breast cancer; CancerResearch, 49:2087.

[0135] Zweig, M. H. and Campbell, G., 1993. Receiver-OperatingCharacteristic (ROC) Plots: A Fundamental Evaluation Tool in ClinicalMedicine; Clinical Chemistry, 39:561-577.

[0136] Other References Related to Subject Matter

[0137] Ali, I., Campbell, G., Lidereau, R., Callahan, R., 1988.Amplification of c-erbB-2 and aggressive human breast tumors; Science,240:1795-1798.

[0138] Allred, D. C., Clark, G. M., Tandon, A. K., Molina, R., Tormey,D. C., Osborne, C. K., Gilchrist, K. W., Mansour, E. G., Abeloff, M.,Eudey, L., McGuire, W. L., 1992a HER-2/neu in node-negative breastcancer: prognostic significance of overexpression influenced by thepresence of in situ carcinoma; Journ. of Clin. Oncology, 10:4:599:605.

[0139] Allred, D. C., Clark, G. M., Molina, R., Tandon, A., Schnitt, S.J., Gilchrist, K. W., Osborne, C. K., Tormey, D. C., McGuire, W. L.,1992b. Overexpression of HER-2/neu and its relationship with otherprognostic factors change during the progression of in situ to invasivebreast cancer; Human Pathology, 23:9:974-979.

[0140] Bacus, S. S., Ruby, S. G., Weinberg, D. S., Chin, D., Ortiz, R.,Bacus, J. W., 1990. HER-2/neu oncogene expression and proliferation inbreast cancers; Amer. Journal of Pathol., 137:1:103-111.

[0141] Battifora, H., Gaffey, M., Esteban, J., Mehta, P., Bailey, A.,Faucett, C., Niland, J., 1991. Immunohistochemical assay of neu/c-erbB-2oncogene product in paraffin-embedded tissues in early breast cancer:retrospective follow-up study of 245 stage I and II cases, ModernPathology, 4:4:466-474.

[0142] Baum, M., Brinkley, D., Dosset, J., 1988. Controlled trial oftamoxifen as a single adjuvant agent in the management of early breastcancer. Analysis at eight years by Nolvadex Adjuvant Trial Organization;British Journal of Cancer, 57:608-611.

[0143] Berns, E. M. J. J., Klijn, J. G. M., Van Staveren, I. L.,Portengen, H., Noordegraaf, E., Foedens, J. A., 1992. Prevalence ofamplification of the oncogenes c-myc, HER2/neu, and int-2 in onethousand human breast tumors: Correlation with steroid receptors; Eur.J. Cancer, 28: 697-700.

[0144] Borg, A., Tandon, A. K., Sigurdsson, I., Clark, G. M., FernöM.,Fuqua, S. A. W, Killander, D., McGuire, W. L., 1990. HER-2/neuamplification predicts poor survival in node-positive breast cancer;Cancer Research, 50:4332-4337.

[0145] Borg, A., Baldetorp, B., Fernö, M., Killander, D., Olsson, H.,Sigurdsson, H., 1991a ERBB2 amplification in breast cancer with a highrate of proliferation; Oncogene, 6:137-143.

[0146] Borg, A., Sigurdsson, H., Clark, G. M., Fernö, M., Fuqua, S. A.W., Olsson, H., Killander, D., McGurie, W. L., 1991 b. Association ofINT2/HST1 coamplification in primary breast cancer withhormone-dependent phenotype and poor prognosis; Br. Journal Cancer,63:1:136-142.

[0147] Bouchard, L., Lamarre, L., Tremblay, P. J., Jolicoeur, P., 1989.Stochastic appearance of mammary tumors in transgenic mice carrying theMMTV/c-neu oncogene; Cell, 57:931-936.

[0148] Breast Cancer Trials Committee, Scottish Cancer Trials Office,Edinburgh, 1987. Adjuvant tamoxifen in the management of operable breastcancer: The Scottish Trial. Lancet, 2, 171-175.

[0149] Callahan, R., Campbell, G., 1989. Mutations in human breastcancer: an overview; J. Natl. Cancer Inst., 81:1780-1786.

[0150] Clark, G. M., McGuire, W. L., 1991. Follow-up study of HER-2/neuamplification in primary breast cancer; Cancer Res, 51:944-948.

[0151] Coussens, L., Yang-Feng, T. L., Liao, Y. C., Chen, E., Gray, A.,McGrath, J., Seeburg, P. H., Libermann, T. A., Schlessinger, J.,Francke, U., Levinson, A., Ulrich, A., 1985. Tyrosine kinase receptorwith extensive homology to EGF receptor shares chromosomal location withneu oncogene; Science, 230:1132-1139.

[0152] Descotes, F., Pavy, J. J., Adessi, G. L., 1993. Human breastcancer: correlation study between HER-2/neu amplification and prognosticfactors in an unselected population; Anticancer Research., 13:1:119-124.

[0153] Donovan-Peluso, M., Contento, A. M., Tobon, H., Ripepi, B.,Locker, J., 1991. Oncogene amplification in breast cancer; Amer. Journalof Pathology, 138:4:835-845.

[0154] Drebin, J. A., Link, V. C., Weinberg, R. A., Greene, M. I., 1986.Inhibition of tumor growth by a monoclonal antibody reactive with anoncogene-encoded tumor antigene; PNAS USA, 83:9129-9133.

[0155] Dressier, L. G., Seamer, L. C., Owens, M. A., Clark, G. M.,McGuire, WL., 1988. DNA flow cytometry and prognostic factors in 1331frozen breast cancer specimens; Cancer, 61:420-427.

[0156] Fallenius, A. G., Auer, G. U., Carstensen, J. M., 1988.Prognostic significance of DNA measurements in 409 consecutive breastcancer patients; Cancer, 62:331-341.

[0157] Fisher, B., Constantino, J., Redmond, C., 1989a. A randomizedclinical trial evaluating tamoxifen in the treatment of patients withnode-negative breast cancer who have estrogen-receptor-positive tumors;New England Journal of Medicine, 320:479-484.

[0158] Fisher, B., Redmond, C., Nikolay, V., 1989b. A randomizedclinical trial evaluating sequential methotrexate and fluorouracil inthe treatment of patients with node-negative breast cancer who haveestrogen-receptor-negative tumors; New England Journal of Medicine,320:473-478.

[0159] Fisher, E. R., Redmond, C., Fisher, B., Bass, G., BSS andcontributing NSABP Investigators, 1990. Pathologic findings from thenational surgical adjuvant breast and bowel projects (NSABP); Cancer,65:2121-2128.

[0160] Fontaine, J., Tesseraux, M., Klein, V., Basieri, G., Blin, N.,1988. Gene amplification and expression of the neu (c-erbB-2) sequencein human mammary carcinoma; Oncology (15)45:360-363.

[0161] Fukushige, S. I., Matsubara, K. I., Yoshida, M., et al., 1986.Localization of a novel v-erbB-related gene, c-erbB-2, on humanchromosome 17 and its amplification in a gastric cancer cell line; Mol.Cell Biol., 6:955-958.

[0162] Giampietro, G., Gullick, W. J., Bevilacqua, P., Sainsbury, R. C.,Meli, S., Boracchi, P., Testolin, A., La Malfa, G., Pozza, F., 1992.Human Breast Cancer: Prognostic significance of the c-erbB-2 oncoproteincompared with epidermal growth factor receptor, DNA Ploidy, andconventional pathologic features; Journal of Clinical Oncol.,10:686-695.

[0163] Gullick, W. J., Love, S. B., Wright, C., Barnes, D. M.,Gusterson, B., Harris, A. L., Altman, D. G., 1991. c-erbB-2 proteinoverexpression in breast cancer is a risk factor in patients withinvolved and uninvolved lymph nodes, Br. Journal of Cancer, 63:434-438.

[0164] Gusterson, B. A., Gelber, R. D., Goldhirsch, A., Price, K. N.,Save-Soderbergh, J., Anbazhagan, R., Styles, J., Rudenstam, C. M.,Golouh, R., Reed, R., Martnez-Tello, F., Tiltman, A., Torhorst, J.,Grigolato, P., Beitelheim, R., Neville, A. M., Burki, K., Castiglione,M., Collins, J., Lindtner, J., Senn, H-J., for the International(Ludwig) Breast Cancer Study Group, 1992. Prognostic Importance ofc-erbB-2 expression in breast cancer; Journal of Clinical Oncol., 10:1049-1056.

[0165] Harris, J. R., Lippman, M. E., Veronesi, U., Willett, W., 1992.Breast Cancer; The New England Journal of Medicine, 6:390-398.

[0166] Hillner, B. E., Smith, T. J., 1991. Efficacy and costeffectiveness of adjuvant chemotherapy in women with node-negativebreast cancer, New. England Journal of Medicine, 324:160-168.

[0167] Isola, J., Visakorpi, T., Holli, K., Kallioniemi, O., 1992.Association of overexpression of tumor suppressor protein p53 with rapidcell proliferation and poor prognosis in node-negative breast cancerpatients; Journal National Cancer Inst., 84:14:1119-1114.

[0168] Kallioniemi, O. P., Holli, K., Visakorpi, T., Helin, H. H.,Isola, J. J., 1991. Association of c-erB-2 protein over-expression withhigh rate of cell proliferation, increased risk of visceral metastasisand poor long-term survival in breast cancer; Int. Journal Cancer, 49:5:650-655.

[0169] King, C. R., Kraus, M. H., Aaronson, S. A., 1985. Amplificationof a novel v-erbB-related gene in a human mammary carcinoma; Science,229:974-976.

[0170] Kraus, M. H., Popescu, N. C., Amsbaugh, S. C., King C. R., 1987.Overexpression of EGF Receptor-related proto-oncogene erbB-2 in humanmammary tumor cell lines by different molecular mechanisms; EMBOJournal, 6:605-610.

[0171] Kury, F., Sliutz, G., Schemper, M., Reiner, G., Reiner, A.,Jakesz, R., Wrba, F., Zeillinger, R., Knogler, W., Huber, J., Holzner,H., Spona, J., 1990. HER-2 oncogene amplification and overall survivalof breast carcinoma patients; European Journal of Cancer, 26:9:946-949.

[0172] Liu, E., Thor, A., He, M., Barcos, M., Ljung, B. M., Benz, C.,1992. The HER-2 (c-erbB-2) oncogene is frequently amplified in situcarcinomas of the breast; Oncogene, 7:5:1027-32.

[0173] Lovekin, C., Ellis, I. O., Locker, A., Robertson, J. F. R., Bell,J., Nicholson, R., Gullick, W. J., Elston, C. W., Blarney, R. W., 1991.c-erbB-2 oncoprotein expression in primary and advanced breast cancer;British Journal of Cancer, 63:439-443.

[0174] Maguire, H. C., Hellman, M. E., Greene, M. I., Yeh, I., 1992.Expression of c-erbB-2 in situ and in adjacent invasive ductaladenocarcinomas of the female breast; Pathobiology, 60:3:117-121.

[0175] Malkin, D., Li, F. P., Strong, L. C., Fraumeni, Jr., J. F.,Nelson, C. E., Kim, D. H., Kassel, J., Gryka, M. A., Bischoff, F. Z.,Tainsky, M. A., Friend, S. H., 1990. Germ line p53 mutations in afamilial syndrome of breast cancer, sarcomas, and other neoplasms,Science, 250:1233-1238.

[0176] Mansour, E., Gray, R., Shatila, A., 1989. Efficacy of adjuvantchemotherapy in high risk node-negative breast cancer; New EnglandJournal of Medicine, 320:485-490.

[0177] McCann, A. H., Dervan, P. A., O'Regan, M., Codd, M. B., Gullick,W. J., Tobin, B. M., Carney, D. N., 1991. Prognostic significance ofc-erbB-2 and estrogen receptor status in human breast cancer; CancerRes., 51:3296-3303.

[0178] McGuire, W., 1989a Adjuvant therapy of node-negative breastcancer; New England Journal of Medicine, 320:525-527.

[0179] McGuire, W., 1989b. Adjuvant therapy of node-negative breastcancer: another point of view; Journal of the National Cancer Institute,80:1075-1076.

[0180] McGuire, W. L., Clark, G., 1992. Prognostic factors and treatmentdecisions in axillary-node-negative breast cancer; New England Journalof Medicine, 326:26:1756-1761.

[0181] Molina, R., Ciocca, D. R., Tandon, A. K., Allred, D. C., Clark,G. M., Chamness, G. C., Gullick, W. J., McGuire, W. L., 1992. Expressionof HER-2/neu oncoprotein in human breast cancer: a comparison ofimmunohistochemical and western blot techniques; Anticancer Research.,12:1965-1972.

[0182] Muller, W. J., Sinn, E., Pattengale, P. K., et al., 1988.Single-step induction of mammary adenocarcinoma in transgenic micebearing the activated c-neu oncogene; Cell, 54:105-111.

[0183] Neville, A. M., 1991. Detection of tumor antigens with monoclonalantibodies: immunopathology and immunodiagnosis, Curr Opin. Immunol.,3:674-678.

[0184] NIH Consensus Development Panel. Consensus statement: treatmentof early-stage breast cancer. In: Consensus development conference onthe treatment of early-stage breast cancer. Journal of the NationalCancer Institute monographs, No. 11., Washington, D.C., GovernmentPrinting Office, 1-5, 1992 (NIH publication no. 903187).

[0185] O'Reilly, S. M., Barnes, D. M., Camplejohn, R. S., Bartkova, J.,Gregory, W. M., Richards, M. A., 1991. The relationship between c-erbB-2expression, S-phase fraction and prognosis in breast cancer; Br. J.Cancer, 63:3:444-446.

[0186] Page, D. L., 1991. Prognosis and breast cancer; The American.Journal of Surgical Path., 15:4:334-349.

[0187] Paik, S., Hazan, R., Fisher, E., Sass, R. E., Fisher, B., Redmon,C., Schlessinger, J., Lippman, M. E., King, C. R., 1990. Pathologicalfindings from the National Surgical Adjuvant Breast and Bowel Project:prognostic significance of erbB-2 protein overexpression in primarybreast cancer; Journal of Clinical Oncology, 8:103-112.

[0188] Patterson, M. C., Dietrich, K. D., Danyluk, J., Paterson, A. H.G., Lees, A. W., Jamil, N., Hanson, J., Jenkins, H., Krause, B. E.,McBlain, W. A., Slamon, D. J., Fourney, R. M., 1991. Correlation betweenc-erbB-2 amplification and risk of recurrent disease in node-negativebreast cancer; Cancer Research, 51:556-567.

[0189] Pauletti, G., Godolphin, W., Press, M. F., Slamon, D. J. 1996.Detection and quantitation of HER-2/neu gene amplification in humanbreast cancer archival material using fluorescence in situhybridization; Oncogene, Jul. 4;13(1):63-72.

[0190] Perren, T. J., 1991. c-erbB-2 oncogene as a prognostic marker inbreast cancer; British Journal of Cancer; 63:328-332.

[0191] Persons, D. L., Borelli, K. A., Hsu, P. H. 1997. Quantitation ofHER-2/neu and c-myc gene amplification in breast carcinoma usingfluorescence in situ hybridization; Mod Pathol, Jul.; 10(7):720-727.

[0192] Press, M. F., Bernstein, L., Thomas, P. A., Meisner, L. F., Zhou,J. Y., Ma, Y., Hung, G., Robinson, R. A., Harris, C., El-Naggar, A.,Slamon, D. J., Phillips, R. N., Ross, J. S., Wolman, S. R., Flom, K. J.,1997. HER-2/neu gene amplification characterized by fluorescence in situhybridization: poor prognosis in node-negative breast carcinomas;Journal of Clinical Oncol, Aug. 15(8):2894-2904.

[0193] Press, M., Cordon-Carlo, C. and Slamon, D., 1990. Expression ofthe HER-2/neu protooncogene in normal adult and fetal tissues; Oncogene,5: 953-962.

[0194] Press, M. F., Pike, M. C., Chazin, V. R., Hung, G., Udove, J. A.,Markowicz, M., Danyluk, J., Godolphi W., Sliwkowski, M., Akita, R.,Paterson, M. C., Slamon, D. J., 1993. HER-2/neu Expression inNode-negative Breast Cancer: Direct Tissue Quantitation by ComputerizedImage Analysis and Association of Overexpression with Increased Risk ofRecurrent Disease; Cancer Research, 53:4960-4970.

[0195] Rhein R., 1993. Early study results of antibodies targeting HER-2proteins seen as promising; Cancer and Genetics Report, pages 8-9.

[0196] Richner, J., Gerber, H. A., Locher, G. W., Goldhirsch, A.,Gelber, R. D., Gullick, W. J., Berger, M. S., Groner, B., Hynes, N. E.,1990. c-erb B-2 protein expression in node negative breast cancer;Annals Oncol., 1: 263-268.

[0197] Rilke, F., Colnaghi, M. I., Cascinelli, N., Andreola, S.,Baldini, M. T., Bufalino, R., Porta, G. D., Menard, S., Pierotti, M. A.,Testori, A., 1991. Prognostic significance of HER-2/neu expression inbreast cancer and its relationship to other prognostic factors; Int. J.Cancer, 49:44-49.

[0198] Rodriguez, G. C., Boente, M. P., Berchuck, A., Whitaker, R. S.,O'Briant, K. C., Xu, F., Bast, R. C., 1993. The effect of antibodies andimmunotoxins reactive wit ER-2/neu on growth of ovarian and breastcancer cell lines; Am. J. Obstet Gynecol, 168:1:1:228-232.

[0199] Rosai, J., 1991. Borderline epithelial lesions of the breast;Amer. J. Surg. Path., 15:209-221.

[0200] Ross, J. S., Nazeer, T., Church, K., Amato, C., Figge, H.,Rifkin, M. D., Fisher, A. G., 1993. Contribution of HER-2/neu OncogeneExpression to Tumor Grade and DNA Content Analysis in the Prediction ofProstatic Carcinoma Metastasis; Cancer, 72:3020-3028.

[0201] Schimmelpenning, H., Eriksson, E. T., Falkmer, U. G., Azavedo,E., Svane, G., Auer, G. U., 1992. Expression of the c-erbB-2proto-oncogene product and nuclear DNA content in benign and malignanthuman breast parenchyma; Virchows Arch. A Pathol. Anat Histopathol,420:5:433-440.

[0202] Singleton, T. P., Stickler, J. G., 1992. Clinical and pathologicsignificance of the c-erbB-2 (HER-2/neu) oncogene; Pathol. Annu.,27:165-190.

[0203] Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich,A., McGuire, W. L., 1987. Human breast cancer: correlation of relapseand survival with amplication of the HER-2/neu oncogene; Science, Jan.9;235(4785):177-182.

[0204] Slamon, D. J., Godolphin, W., Jones, L., Holt, J., Wong. S.,Keith, D., Levin, W., Stuart, S., Udove, J., Ullrich and Press, M.,1989a Studies of the HER-2/neu proto-oncogene in human breast andovarian cancer; Science, 244:707-712.

[0205] Slamon, D. J., Press, M. F., Godolphin, W., Ramos, L., Haran, P.,Shek, L., Stuart, S. G., Ullrich, A., 1989b. Studies of the HER-2/neuproto-oncogene in human breast cancer; Cancer Cells, 7:371-384.

[0206] Tandon, A. K., Clark, G. M., Chamness, G. C., et al., 1989.HER-2/neu oncogene protein and prognosis in breast cancer; J. Clin.Oncol., 7:1120-1128.

[0207] Thor, A. D., Schwartz, L. H., Koerner, F. C., et al., 1989.Analysis of c-erbB-2 expression in breast carcinomas with clinicalfollow-up; Cancer Res., 49:7147-7152.

[0208] Tiwari, R., Borgen, P. I., Wong, G. Y., Cordon-cardo, C.,Osborne, M. P., 1992. HER-2/neu amplification and overexpression inprimary human breast cancer is associated with early metastasis;Anticancer Research, 12:419-426.

[0209] Tommasi, S., Giannella, C., Paradiso, A., Barletta, A., Mangia,A., Simone, G., Primavera, A. T., Albarani, V., Schittulli, F., Longo,S., 1992. HER-2/neu gene in primary and local metastatic axillary lymphnodes in human breast tumors; Int. J. Biol. Markers, 7:2:107-113.

[0210] Tsuda, H., Hirohashi, S., Shimosato, Y., Hirota, T., Tsugane, S.,Watanabe, S., Terada, M., Yamamoto, H., 1990. Correlation betweenhistologic grade of malignancy and copy number of c-erbB-2 gene inbreast carcinoma. A retrospective analysis of 176 cases; Cancer,65:1794-1800.

[0211] Van de Vijver, M., van de Bersselaar, R., Devilee, P., et al,1987. Amplification of the neu (c-erbB-2) oncogene in human mammarytumors is relatively frequent and is often accompanied by amplificationof the linked c-erbA oncogene; Mol. Cell. Biol., 7:2019-2023.

[0212] Winstanley, J., Cooke, T., George, W. D., Murray, G., Holt, S.,Croton, R., Griffiths, K., Nicholson, R., 1991. The long term prognosticsignificance of oestrogen receptor analysis in early carcinoma of thebreast; British J. Cancer, 64:99-101.

[0213] Wolman, S. R., Henderson, A. S., 1989. Chromosomal aberrations asmarkers of oncogene amplification.; Human Pathology, 20:308-315.

[0214] Xing, W. R., Gilchrist, K. W., Harris, C. P., Samson, W.,Meisner, L. F. 1996. FISH detection of HER-2/neu oncogene amplificationin early onset breast cancer, Breast Cancer ResearchTreat;39(2):203-212.

[0215] Xu, F., Lupu, R., Rodriguez, G. C., Whitaker, R. S., Boente M.P., Berchuck, A., Yu, Y., Desombre, K. A., Boyer, C. M., Bast, R. C.,1993. Antibody-induced growth inhibition is mediated throughimmunochemically and functionally distinct epitopes on the extracellulardomain of the c-erbB-2 (HER-2/neu) gene product; Int. J. Cancer,53:3:401-408.

[0216] Zhou, D., Ahuja, H. and Cline, M., 1989. Proto-oncogeneabnormalities in human breast cancer: c-ERB-2 amplification does notcorrelate with recurrence of disease; Oncogene, 4:105-108.

[0217] All references mentioned above are herein incorporated byreference.

We claim:
 1. A method for determining the likelihood of cancerrecurrence in a patient comprising the steps of: (a) obtaining tumorcells from the patient; (b) applying to each of said cells a detectableprobe capable of hybridizing to the HER-2/neu gene; (c) counting thenumber of HER-2/neu genes per cell; and (d) determining the likelihoodof cancer recurrence wherein an average of about 10 or more HER-2/neugenes per tumor cell indicates a high likelihood of cancer recurrenceand an average of about 3 or fewer BER-2/neu genes per tumor cellindicates a low likelihood of cancer recurrence.
 2. The method accordingto claim 1 wherein a control cell line is substantially simultaneouslytested.
 3. The method according to claim 1 wherein the number of cellscounted is about
 40. 4. The method according to claim 1 wherein thenumber of cells counted is about
 20. 5. A method for determining thelikelihood of cancer recurrence in a patient comprising the steps of:(I) obtaining tumor cells from the patient; (ii) applying to said cellsa detectable probe capable of hybridizing to the HER-2/neu gene; (iii)counting the number of HER-2/neu genes in cells; and (iv) determiningthe likelihood of cancer recurrence wherein an average of at least about4 HER-2/neu genes per tumor cell indicates a high likelihood of cancerrecurrence.
 6. The method according to claim 5 wherein at least onecontrol cell line is substantially simultaneously tested.
 7. The methodaccording to claim 5 wherein the number of cells with HER-2/neu genescounted therein is no more than about
 20. 8. A kit of quality controlcell lines for a HER-2/neu gene amplification detection systemcomprising: (i) a non-amplified control cell line that exhibits a meanof about 3 or fewer HER-2/neu genes per cell; and (ii) an amplifiedcontrol cell line that exhibits a mean of about 10 or more HER-2/neugenes per cell.
 9. The kit according to claim 8 and further comprising alow amplified control cell line that exhibits a mean of between about 3and 10 HER-2/neu genes per cell.
 10. A kit of quality control cell linesfor use with a breast cancer gene amplification detection systemaccording to claim 9 comprising: (I) ATCC HTB 30 (SK-BR-3) cell line;(ii) ATCC HTB 132 (MDA-MB-468) cell line; and (iii) ATCC HTB 133 (T-47D)cell line.
 11. A kit for determining the number of copies of HER-2/neugenes in a cell comprising; a) a detectable probe capable of hybridizingto the HER-2/neu gene; and b) at least one control cell line selectedfrom the group consisting of one that exhibits a mean of about 10 ormore HER-2/neu genes per cell, one that exhibits a mean of between about3 and 10 HER-2/neu genes per cell, and one that exhibits a mean of about3 or fewer HER-2/neu genes per cell.
 12. The kit according to claim 11further comprising at least two of the control cell lines.
 13. The kitaccording to claim 12 further comprising all three of the control celllines.
 14. A method for preparing a standardized control slidecomprising mixing cells of a cell line in a liquid to form a suspension,solidifying the suspension, whereby the cells are dispersed throughoutthe suspension to produce a solid form, cutting thin sections from thesolid form, and mounting the thin section onto a slide.
 15. The methodaccording to claim 14 further comprising embedding the solid form in asolid matrix.
 16. The method according to claim 15 wherein the solidmatrix is paraffin.
 17. The method according to claim 14 furthercomprising staining the section.
 18. The method according to claim 14further comprising preparing the section for binding an added ligand toa receptor in or on the cells.
 29. The method according to claim 14further comprising preparing the section so that nucleic acid in thecells is capable of hybridizing to an added solution containing acomplementary nucleic acid probe.
 20. A quality control material for useas a standardized control comprising: (a) a first cell line having anamplified HER-2/neu gene copy number; and (b) a second cell line havinga non-amplified HER-2/neu copy number; wherein all of the cells in thequality control material are distributed throughout a solid medium inthree dimensions.
 21. A thin section of a quality control materialconsisting essentially of a thin section cut from the material of claim20.